Prolonged α-amanitin treatment of cells for studying mutated polymerases causes degradation of DSIF160 and other proteins

Tsao, David C.; Park, Noh Jin; Nag, Anita; Martinson, Harold G.

doi:10.1261/rna.030452.111

Cited by 8 publications

(6 citation statements)

References 32 publications

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“…We took advantage of an Rpb1 mutation that confers resistance to α-amanitin. Although α-amanitin has been reported to affect the level of short-lived proteins, and therefore indirectly impair pre-mRNA splicing (44), our results suggest that the splicing machinery is intact in our experimental conditions. Indeed, no splicing defect was observed when pre-mRNAs were synthesized by the α-amanitin–resistant wild-type Rpb1.…”

Section: Discussionmentioning

confidence: 53%

CTD serine-2 plays a critical role in splicing and termination factor recruitment to RNA polymerase II in vivo

Eick

Bensaude

2012

Nucleic Acids Research

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Co-transcriptional pre-mRNA processing relies on reversible phosphorylation of the carboxyl-terminal domain (CTD) of Rpb1, the largest subunit of RNA polymerase II (RNAP II). In this study, we replaced in live cells the endogenous Rpb1 by S2A Rpb1, where the second serines (Ser2) in the CTD heptapeptide repeats were switched to alanines, to prevent phosphorylation. Although slower, S2A RNAP II was able to transcribe. However, it failed to recruit splicing components such as U2AF65 and U2 snRNA to transcription sites, although the recruitment of U1 snRNA was not affected. As a consequence, co-transcriptional splicing was impaired. Interestingly, the magnitude of the S2A RNAP II splicing defect was promoter dependent. In addition, S2A RNAP II showed an impaired recruitment of the cleavage factor PCF11 to pre-mRNA and a defect in 3′-end RNA cleavage. These results suggest that CTD Ser2 plays critical roles in co-transcriptional pre-mRNA maturation in vivo: It likely recruits U2AF65 to ensure an efficient co-transcriptional splicing and facilitates the recruitment of pre-mRNA 3′-end processing factors to enhance 3′-end cleavage.

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Section: Discussionmentioning

confidence: 53%

CTD serine-2 plays a critical role in splicing and termination factor recruitment to RNA polymerase II in vivo

Eick

Bensaude

2012

Nucleic Acids Research

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“…Thus, while stably transformed cells expressing a-amanitinresistant Rpb1 avoid the problems of transient transfection, such cells initially grow slowly with reduced viability in the presence of a-amanitin (Meininghaus et al 2000;Chapman et al 2004Chapman et al , 2005, indicating that they must undergo certain changes to survive. Indeed, 24-h a-amanitin treatment was recently shown to bring about accelerated degradation of several proteins, including the transcription elongation factor DSIF, and this may complicate interpretation of experimental results (Tsao et al 2012). …”

Section: End Processingmentioning

confidence: 99%

The RNA polymerase II CTD coordinates transcription and RNA processing

2012

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The C-terminal domain (CTD) of the RNA polymerase II largest subunit consists of multiple heptad repeats (consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), varying in number from 26 in yeast to 52 in vertebrates. The CTD functions to help couple transcription and processing of the nascent RNA and also plays roles in transcription elongation and termination. The CTD is subject to extensive post-translational modification, most notably phosphorylation, during the transcription cycle, which modulates its activities in the above processes. Therefore, understanding the nature of CTD modifications, including how they function and how they are regulated, is essential to understanding the mechanisms that control gene expression. While the significance of phosphorylation of Ser2 and Ser5 residues has been studied and appreciated for some time, several additional modifications have more recently been added to the CTD repertoire, and insight into their function has begun to emerge. Here, we review findings regarding modification and function of the CTD, highlighting the important role this unique domain plays in coordinating gene activity.Eukaryotes have three nuclear DNA-dependent RNA polymerases (RNAPs): RNAP I, RNAP II, and RNAP III. RNAP II, responsible for the synthesis of all mRNA as well as many noncoding RNAs (ncRNAs), consists of 12 polypeptides, of which Rpb1, which possesses the enzyme's catalytic activity, is the largest. Rpb1 also contains a C-terminal domain (CTD) composed of tandem heptad repeats that constitutes a unique feature of RNAP II and distinguishes it from all other polymerases. The CTD is conserved from fungi to humans, although the number of repeats and their deviation from the consensus vary, reflecting to a large degree the complexity of the organism. The CTD plays important roles at all steps of the transcription process, including enhancing or modulating the efficiency of all of the RNA processing reactions required for completion of synthesis of the mature RNA. The phosphorylation state of the CTD is critical in determining its activity.

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“…Average speed for these RNA Pol II mutants is 0.5 Kb/min or even slower for the slow mutants (R749H and H1108Y, respectively) and 1.9 Kb/min for the fast mutant as compared to 1.7 Kb/min for the human α‐amanitin‐resistant WT pol II (Fong et al , 2014 ). Nevertheless, prolonged α‐amanitin treatment has been shown to induce accelerated degradation of several proteins including the elongation factor DSIF (Tsao et al , 2012 ). Measurements of RNA Pol II speed obtained using α‐amanitin‐resistant RNA Pol II mutants should thus be taken with care.…”

Section: Rna Pol II Speed Controls Many Co‐transcriptional Processes and Thus Affects The Composition Of The Transcriptomementioning

confidence: 99%

RNA polymerase II speed: a key player in controlling and adapting transcriptome composition

2021

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RNA polymerase II (RNA Pol II) speed or elongation rate, i.e., the number of nucleotides synthesized per unit of time, is a major determinant of transcriptome composition. It controls co‐transcriptional processes such as splicing, polyadenylation, and transcription termination, thus regulating the production of alternative splice variants, circular RNAs, alternatively polyadenylated transcripts, or read‐through transcripts. RNA Pol II speed itself is regulated in response to intra‐ and extra‐cellular stimuli and can in turn affect the transcriptome composition in response to these stimuli. Evidence points to a potentially important role of transcriptome composition modification through RNA Pol II speed regulation for adaptation of cells to a changing environment, thus pointing to a function of RNA Pol II speed regulation in cellular physiology. Analyzing RNA Pol II speed dynamics may therefore be central to fully understand the regulation of physiological processes, such as the development of multicellular organisms. Recent findings also raise the possibility that RNA Pol II speed deregulation can be detrimental and participate in disease progression. Here, we review initial and current approaches to measure RNA Pol II speed, as well as providing an overview of the factors controlling speed and the co‐transcriptional processes which are affected. Finally, we discuss the role of RNA Pol II speed regulation in cell physiology.

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Prolonged α-amanitin treatment of cells for studying mutated polymerases causes degradation of DSIF160 and other proteins

Cited by 8 publications

References 32 publications

CTD serine-2 plays a critical role in splicing and termination factor recruitment to RNA polymerase II in vivo

CTD serine-2 plays a critical role in splicing and termination factor recruitment to RNA polymerase II in vivo

The RNA polymerase II CTD coordinates transcription and RNA processing

RNA polymerase II speed: a key player in controlling and adapting transcriptome composition

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